Prenatal diagnosis of vitamin D-dependent rickets, type II: response to 1,25-dihydroxyvitamin D in amniotic fluid cells and fetal tissues.

0021-972X/90/7104-0937$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society

Vol. 71, No. 4 Printed in U.S.A.

Prenatal Diagnosis of Vitamin D-Dependent Rickets, Type II: Response to 1,25-Dihydroxyvitamin D in Amniotic Fluid Cells and Fetal Tissues YOSEF WEISMAN, NIVA JACCARD, CYRIL LEGUM, ZVI SPIRER, GIDEON YEDWAB, LEA EVEN, SAMUEL EDELSTEIN, ALVIN M. KAYE, AND ZEEV HOCHBERG Bone Disease Unit, Ichilov Hospital, Sackler Faculty of Medicine, Tel Aviv University (Y.W., N.J., C.L., Z.S., G.Y.), Tel Aviv; the Departments of Biochemistry and Hormone Research, the Weizmann Institute of Science (S.E., A.M.K.), Rehovot; and the Departments of Pediatrics, Rambam and Haifa Medical Centers (L.E., Z.H.), Haifa, Israel

of a child with VDDR-II were unable to bind [3H]1,25-(OH)2D3, and the hormone failed to stimulate 24-hydroxylase activity. VDDR-II in this fetus was confirmed after termination of pregnancy by the total inability of 1,25-(OH)2D3 to stimulate 24hydroxylase activity in tissue explants and cell cultures prepared from the fetus's kidney and skin. In contrast, tissues from dead control fetuses responded to stimulation by 1,25-(OH)2D3 with a 3- to 10-fold increase in 24-hydroxylase activity. Fetal kidney and skin explants and cell cultures also synthesized a [3H]l,25-(OH)2D3-like metabolite from [3H]25-OHD3 as early as the 17th week of gestation. 1,25-(OH)2D3 (10 nM) decreased the in vitro synthesis of the [3H]l,25-(OH)2D3-like metabolite in tissues from dead control fetuses, but not from the affected fetus. Thus, human fetuses at midgestation already have the regulatory mechanisms responsive to 1,25-(OH)2D3 present postnatally. The prenatal diagnosis of VDDR-II is now possible and is indicated in a high risk family. {J Clin Endocrinol Metab 71: 937-943, 1990)

ABSTRACT. Vitamin D-dependent rickets type II (VDDR-II; hereditary resistance to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]), an autosomal recessive genetic disease that results from a failure to respond to 1,25-(OH)2D3, is characterized by severe rickets, hypocalcemia, growth retardation, and high prevalence of alopecia. We used amniotic fluid cells in the 17th week of gestation to detect VDDR-II in fetuses at risk for the defect. First, we demonstrated in cells obtained from 15 control pregnancies the presence of a specific high affinity 1,25-(OH)2D3 receptor (Kd = 0.3 x 10~n mol/L; maximal number of binding sites, 6.1 fmol/ mg protein) and l,25-(OH)2D3-induced 25-hydroxyvitamin D324-hydroxylase activity (up to 30-fold increase). Amniotic fluid cells from a woman who had already given birth to a child with VDDR-II contained receptors that bound [3H]1,25-(OH)2D3 normally and responded to 1,25-(OH)2D3 stimulation with a 10-fold increase in 24-hydroxylase activity. The fetus was, therefore, judged unaffected, and a normal baby girl was born. At the age of 16 months she did not demonstrate clinical or biochemical features of VDDR-II. Amniotic fluid cells from another mother

V

administration of very high doses of vitamin D metabolites (1, 6-9). Other patients, including those from families reported in the current study, responded only to long term intracaval calcium infusion therapy (10, 11). Since resistance to 1,25-(OH)2D3 may result in dwarfism with severe skeletal deformations or even death (8, 12), we wished to develop a method for prenatal detection of VDDR-II in pregnant women who had already given birth to a child with such a defect. Human skin fibroblasts contain receptors for 1,25-(OH)2D3 (13, 14) and respond to 1,25-(OH)2D3 stimulation by increased 25hydroxyvitamin D3-24-hydroxylase activity (15-17). In contrast, cultured skin fibroblasts from patients with VDDR-II contain defective 1,25-(OH)2D3 receptors, and their lack of biological response to 1,25-(OH)2D3 is shown by the total inability of the hormone to stimulate 24hydroxylase activity in these cells (15-17).

ITAMIN D-dependent rickets type II (VDDR-II; hereditary resistance to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]) is an autosomal recessive genetic disease resulting from a failure to respond to 1,25-(OH)2D3. The characteristic clinical features of this disease are severe rickets, hypocalcemia, growth retardation, high prevalence of alopecia, and elevated circulating levels of 1,25(OH)2D3 (1-3). The molecular basis of the disease is heterogeneous and includes defects in the 1,25-(OH)2D3 receptor, which cause impaired hormone-receptor binding or abnormal binding of the receptor to DNA (4, 5). Therapy for VDDR-II is difficult. Some patients have shown biochemical and clinical improvement after the Received September 5,1989. Address all correspondence and requests for reprints to: Y. Weisman, M.D., Bone Disease Unit, Ichilov Hospital, 6 Weizman Street, Tel Aviv 64239, Israel.

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Since the fetal epidermis is a major source of amniotic fluid fibroblast-like cells (18), it is reasonable to expect that analysis of [3H]1,25-(OH)2D3 binding and 1,25(OH)2D3-stimulated 24-hydroxylase activity in amniotic fluid cells at midgestation could be used for prenatal diagnosis of VDDR-II. In this paper we describe the prenatal detection of the presence and absence of resistance to 1,25-(OH)2D3 in two fetuses at risk for VDDR-II as well as data on vitamin D metabolism and responsiveness to 1,25-(OH)2D3 in human fetal tissue at midgestation.

Subjects and Methods Subjects Case 1. A 26-yr-old Moslem-Arab woman is the mother of a 6-

yr-old boy with VDDR-II. Details of the boy's clinical and biochemical features, including receptor studies, were documented in previous reports (11, 19). Since May 1985, he has been on long term intracaval calcium infusion therapy, which resulted in healing of rickets-osteomalacia and accelerated his growth rate (11). Case 2. A 24-yr-old Moslem-Arab woman is the mother of a 3yr-old girl with VDDR-II. This patient is the first cousin to case 1 and three previously reported children with VDDR-II (3). The girl demonstrated rickets and alopecia as early as her third month of life. Extracts of the girl's cultured skin fibroblasts failed to bind [3H]1,25-(OH)2D3. She is responding favorably to a program of long term intracaval calcium infusion therapy. In both of the above cases the parents were anxious to have another child and asked for genetic counseling. Informed consent for the procedure and study was obtained from both families. An amniotic fluid sample was obtained by transabdominal amniocentesis at the 17th week of gestation in both women. Controls. Amniotic fluid samples were obtained from 15 normal women in their 17th week of gestation for routine diagnosis of chromosomal aberrations. Cells that were not used for chromosomal studies were subcultured and used for studies of [3H] 1,25-(OH)2D3 binding and responsiveness to the hormone. Samples of fetal kidney and skin tissues were obtained from two dead fetuses after legal termination of pregnancy at the 17th and 22nd weeks of gestation (approved by the Tel-Aviv Sourasky Medical Center Committee on Termination of Pregnancy). The use of the fetal tissues was discussed and approved by the pregnant women after the committee's decision to abort had been made. Fetal tissues (treated with the same respect given to human cadavers) were used to prepare tissue explants and cell cultures. Cell cultures Amniotic fluid samples (10-20 ml) were centrifuged at 800 X g for 10 min to obtain cells, which were cultured in medium 199 (M-199) supplemented with 20% fetal calf serum, gluta-

JCE & M • 1990 Vol 71 • No 4

mine (2 mM), penicillin (100 U/mL), streptomycin (50 mg/ mL), and mycostatin (12 U/mL). After establishment of cell cultures, monolayers were subcultured in M-199 supplemented with 10% fetal calf serum and used at passages 2-4. Cultures of fetal skin fibroblasts were established from fibroblast outgrowths of minced skin fragments maintained in M-199 supplemented with 20% serum. Monolayers were then subcultured in medium supplemented with 20% fetal calf serum. Kidney cells cultures were established from trypsinized fetal kidney tissue. The cells were cultured in BGJb medium supplemented with 10% fetal calf serum and antibiotics. Fetal kidney and skin tissues were cut into small pieces (2 mm in any dimension; 100 mg/dish) and used directly in explant experiments. Binding studies 1,25-(OH)2D3 receptor assays were performed by previously described methods (13, 15). In brief, cells were incubated with serum-free medium for 16 h and then washed twice with cold phosphate-buffered saline and homogenized in hypertonic buffer (300 mM KC1, 10 mM Tris-HCl, 1.5 mM EDTA, 5 mM dithiothreitol, and 10 mM sodium molybdate, pH 7.4). The hypertonic buffer extracts receptors previously referred to as cytosol and nuclear. The homogenates were centrifuged at 100,000 x g for 1 h, and samples of the supernatant extracts were incubated with various concentrations (0.025-0.5 mM) of [3H]1,25-(OH)2D3 (180 Ci/mmol; Amersham, Bucks, England) for 3 h at 20 C. Nonspecific binding was assessed in parallel by the addition of a 200-fold concentration of nonradioactive 1,25(OH)2D3. Bound and free 1,25-(OH)2D3 were separated by the dextran-coated charcoal method. Sucrose gradient analysis Samples of cell extracts were incubated with 0.8 nM [3H] 1,25-(OH)2D3 with and without a 100-fold excess of unlabeled 1,25-(OH)2D3 for 2 h at 20 C. After separation of the free metabolite by dextran-coated charcoal, 300 fxh labeled extract were layered on top of a 4.7-mL 5-20% sucrose gradient, prepared in hypertonic buffer, and centrifuged for 18 h at 260,000 x g at 4 C. Fractions were collected and measured for radioactivity. Extracts prepared from the intestinal mucosa of vitamin D-deficient chicks were analyzed under the same conditions as a comparison. Ovalbumin (Sigma, St. Louis, MO) was used as a standard (sedimentation coefficient, 3.6S). 24-Hydroxylase and la-hydroxylase assays Assays were performed essentially as previously described (15-17). Confluent monolayers of amniotic fluid cells, fetal skin fibroblasts, or kidney cells cultured in medium containing 1% serum were treated with 1,25-(OH)2D3 (10 nM) in ethanol or with ethanol alone. After 16 h of incubation, cells were rinsed twice with medium, removed by trypsinization, rewashed, and suspended in medium containing 1% serum and 20 mmol/L HEPES. Samples of cell suspensions (106 cells/1 mL) were incubated for 1 h at 37 C with 100 pmol [3H]25OHD3 (19.2 Ci/ mmol; Amersham). The reaction was terminated, and the metabolites were extracted by the addition of chloroform-methanol (2:1, vol/vol). Explants of fetal kidney and skin were also


PRENATAL DIAGNOSIS OF VDDR-II assayed for 24- and la-hydroxylase activities. Explants (2 mm in any dimension; 100 mg/dish) were incubated in M-199 in the presence or absence of unlabeled 1,25-(OH)2D3 (10 nM) for 16 h at 37 C. The explants were then washed twice with medium and incubated with 1 mL medium containing 1% serum with 100 pmol [3H]25OHD3 for 3 h. The explants and medium were extracted with chloroform-methanol (2:1, vol/vol). Authentic unlabeled 24,25-(OH)2D3 was added to the lipid extracts, and the metabolites were separated on Sephadex LH-20 columns (0.7 x 20 cm) eluted with chloroform-petroleum ether (40-60 C; 1:1, vol/vol). The fractions containing [3H]24,25-(OH)2D3 and [3H]1,25-(OH)2D3 were analyzed by high performance liquid chromatography (HPLC) using a ^-Porasil column (Waters Associates, Milford, MA) and hexane-isopropanol (93:7, vol/ vol) as the mobile phase (20). In some experiments the [3H] 1,25-(OH)2D3 fraction was also analyzed by using dichlormethane-isopropanol (19:1, vol/vol) as the mobile phase. The [3H] 24,25-(OH)2D3 fraction was also analyzed by reverse phase HPLC on a Bondapak C18 column with methanol-water (80:20, vol/vol) as the mobile phase. [3H]24,25-(OH)2D3 and [3H]1,25(OH)2D3 were identified by comigration with the unlabeled reference standards, which were monitored by UV absorbance at 254 nm. The 24- and la-hydroxylase activities in cell cultures were expressed as picomoles of [3H]24,25-(OH)2D3 or [3H]1,25(OH)2D3 produced per h/106 cells. The enzyme activity in tissue explants was expressed as picomoles of metabolite produced per h/100 mg wet wt of tissue.

Results Amniotic fluid cell cultures from control women

939 70.*

7-

50-

6205-

10-

4-

0Jr0

40

80

120

1.25 -(0H) 2

160

D 3 (pM)

Kd » O.3 x 10""M N max = 6.1 f mol mg /proiein

2-

I-

oJr

O

10

20

30

40

3

L HJ I,25-(OH) 2 D 3

50

Bound

70

60

( pM)

FIG. 1. Analysis of [3H]1,25-(OH)2D3 binding in extracts of cultured amniotic fluid cells from control women. A, Saturation analysis of specific binding. B, Scatchard plot of specific binding data. See Materials and Methods for experimental details.

Extracts of cultured amniotic fluid cells obtained from

control women were analyzed for 1,25-(OH)2D3 receptor using different concentrations of [3H]1,25-(OH)2D3 (Fig. 1A). The specific binding reached a plateau at approximately 0.1 nM [3H]1,25-(OH)2D3. Scatchard analysis of the binding data (Fig. IB) indicates the presence of high affinity receptors for 1,25-(OH)2D3, with an equilibrium dissociation constant (Kd) for [3H]1,25-(OH)2D3 of 0.3 x 10" u M and a maximum number of binding sites of 6.1 fmol/mg protein. A possible binding site with substantially lower affinity for 1,25-(OH)2D3 was also detected. The specificity of the receptor for 1,25-(OH)2D3 was demonstrated in separate binding experiments (data not shown) in which there was no displacement of [3H]1,25(OH)2D3 when extracts were incubated with a 100-fold excess of either unlabeled 25OHD3 or 24R,25-(OH)2D3. Sedimentation analysis of extracts of amniotic fluid cells labeled with [3H]1,25-(OH)2D3 revealed a single peak of radioactivity sedimenting at 3.5S (Fig. 2) similar to that found for 1,25-(OH)2D3 receptor in chick intestinal mucosa. The binding was abolished with a 200-fold molar excess of unlabeled 1,25-(OH)2D3. The basal rate of [3H]24,25-(OH)2D3 production by 15 cultures of amniotic fluid cells incubated with 100 pmol [3H]25OHD3 averaged less than 0.6 pmol/106 cells-1 h

3 6S

220 200180160140120 10080-

6040200- "!,_,

,

,

29 26 26 24

,

,

,

,

,

,__,

22 20 18 16 14 12 |0 Fraction

,

,

,

8

6

4

,.Bot. 2

Number

FIG. 2. Sucrose gradient analysis of 1,25-(OH)2D3 receptors from amniotic fluid cells of control women (O) and from chick intestinal mucosa (•). Samples of cell extracts were incubated with [3H]1,25-(OH)2D3 with (A) and without a 100-fold excess of unlabeled 1,25-(OH)2D3 for 2 h at 20 C. Bound and free hormones were separated by dextrancoated charcoal, and extracts were layered on 5-20% sucrose gradients made with hypertonic buffer. Ovalbumin (3.6S) was used as a standard.


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JCE & M • 1990 Vol 71 • No 4

TABLE 1. l,25-(OH)2D3-induced 24-hydroxylase activity in amniotic fluid cells from mothers of VDDR-II children [3H]24,25-(OH)2D3 produced (pmol/106 cells -h"1)

5.0-

2.5

o E

1.0

l,25-(OH)2D3-induced activity

Case 1 Control

0.44 ± 0.15 0.54 ± 0.28

4.80 ± 0.62° 6.10 ± 1.67"

Case 2 Control

0.90 ± 0.54 1.02 ± 0.26

0.81 ± 0.34 8.56 ± 1.94"

Cell cultures were treated with 10 nM 1,25-(OH)2D3 for 16 h. Enzyme activity was assayed by measuring conversion of [3H]25OHD3 to [3H] 24,25-(OH)2D3 (15-17). Values are the mean ± SD of triplicate dishes. " Differs from cultures not treated with 1,25-(OH)2D3) P < 0.001.

Q.

X

Basal activity

0.5

o

niotic fluid cells o b t a i n e d from case 2 was found. T h e s e

2. 0.25

cells also did not respond to treatment with 1,25(OH)2D3, as shown by the inability of the hormone to stimulate 24-hydroxylase activity (Table 1).

X

0.10

Responsiveness to 1,25-(OH)2D3 and 25OHD3 metabolism in fetal tissues 0.05

L

No Treatment

25-(OH)rD3 (10 nM)

FlG. 3. The induction of 24-hydroxylase activity by 1,25-(OH)2D3 in amniotic fluid cells obtained from 15 pregnant women at the 16th to 17th weeks of gestation. Cell cultures were treated with 10 nM 1,25(OH)2D3 for 16 h. Enzyme activity was assayed by measuring the conversion of [3H]25OHD3 to [3H]24,25-(OH)2D3 (15-17).

(Fig. 3). When these cell cultures were treated with 10 nM 1,25-(OH)2D3 for 16 h, the production of [3H]24,25(OH)2D3 increased in all cases from 2- to 30-fold (Fig. 3). Cycloheximide (10 yug/mL) caused a significant inhibition of the l,25-(OH)2D3-induced increase in 24-hydroxylase activity (data not shown), indicating that the l,25-(OH)2D3-induced increase in 24-hydroxylase activity was due to de novo protein synthesis. Amniotic fluid cells from mothers of VDDR II children Extracts of cultured amniotic fluid cells obtained from case 1 demonstrated specific binding to [3H]1,25(OH)2D3. Sucrose gradient analysis of these preparations revealed a single radioactive peak at 3.5S, similar to that found in cells obtained from control women. Cells from case 1 also responded with a 10-fold increase in 25OHD324-hydroxylase activity after stimulation by 1,25(OH)2D3 (Table 1). We, therefore, reached the tentative conclusion that the l,25-(OH)2D3-receptor effector system was normal in this fetus. Indeed, a normal baby girl was born at term. At the age of 16 months, she showed no clinical or biochemical features of VDDR-II. No binding of [3H]1,25-(OH)2D3 to extracts of am-

The findings in amniotic fluid cells indicated that the fetus of case 2 lacked a normal 1,25-(OH)2D3 receptoreffector system. Therefore, the pregnancy was terminated at the 22nd week of gestation at the parents' request with the approval of the Tel-Aviv Sourasky Medical Center Committee on Termination of Pregnancy, based on the fact that the woman had one child affected with vitamin D-dependent rickets type II and the findings in amniotic fluid cells, which showed no 1,25-(OH)2D3 receptor and no response to the hormone. The parents gave their consent to the use of the fetal tissue for biochemical studies. The diagnosis of VDDRII in this fetus was confirmed by the total inability of 1,25-(OH)2D3 to stimulate 24-hydroxylase activity in explants of skin and kidney tissue as well as cultures of skin fibroblasts and kidney cells (Fig. 4). Tissue explants and cell cultures prepared from two dead control fetuses responded to stimulation by 1,25-(OH)2D3 with a 3- to 10-fold increase in 24-hydroxylase activity (Fig. 4). Analysis of [3H]25OHD3 metabolism in fetal tissues demonstrated that as early as the 17th week of gestation, fetal kidney and skin synthesized a [3H]1,25-(OH)2D3like metabolite (Fig. 5), which comigrated on two HPLC systems with authentic 1,25-(OH)2D3. 1,25-(OH)2D3 (10 nM) decreased the synthesis of the [3H]1,25-(OH)2D3like metabolite in tissues from dead control fetuses, but not in those from the affected fetus (Fig. 5).

Discussion In the present study we have used cultured amniotic fluid cells for the study of the integrity of the 1,25-


PRENATAL DIAGNOSIS OF VDDR-II 5.0

2

Affected Fetus

Control Fetuses

3.0 2.0

h 1.0

2

0.5

0.25

0.1251-

f

No Treatment

t

1, 25 (OH)2D3 (10 nM)

t

t

No Treatment

1,25-(OH),D3 (10 nM)

FIG. 4. Stimulation by 1,25-(OH)2D3 of 24-hydroxylase activity in explants of skin (O) and kidney tissue (A) and in cultured skin fibroblasts (•) and kidney cells (A) obtained from one VDDR-II and two control dead fetuses. Explants and cell cultures were treated with 10 nM 1,25-(OH)2D3 for 16 h. Enzyme activity was assayed by measuring the conversion of [3H]25OHD3 to [3H]24,25-(OH)2D3 (15-17). Each data point is the mean of two or three determinations. 2.0

Control Fetuses

Affected Fetus

1.0

0.5

So, 8

o 0.1

0.05"-

t

I

t

No

1, 25 (OH)JDJ

No

Treatment

(10 nM)

Treatment

t

1,25(OH)2Dj (10 nM)

FlG. 5. Inhibition by 1,25-(OH)2D3 of the synthesis of a [3H]1,25(OH)2D3-like metabolite in explants of skin (O) and kidney tissue (A) and in cultured skin fibroblasts (•) and kidney cells (A) obtained from one VDDR-II and two control dead fetuses. Explants and cell cultures were treated with 10 nM 1,25-(OH)2D3 for 16 h. Production of the [3H] l,25-(OH)2D3-like metabolite was determined by measuring the conversion of [3H]25OHD3 to the [3H]l,25-(OH)2D3-like metabolite (20, 31). Each data point is the mean of two or three determinations.

(OH)2D3 receptor-effector system in fetal tissues and for prenatal detection of VDDR-II in fetuses at risk for such a defect. We found that amniotic fluid cells at midgestation contains specific high affinity 1,25-(OH)2D3 receptors similar to those found in human cultured skin fibro-

941

blasts and other responsive tissues (21). Amniotic fluid cells also responded to 1,25-(OH)2D3 stimulation by increased 25OHD3-24-hydroxylase activity, a sensitive marker for 1,25-(OH)2D3 action (15-17, 22). These findings are in agreement with those of Delvin et al. (23) in six amniotic fluid cell cultures. The possibility of using chorionic villi samples for earlier prenatal diagnosis was explored. However, no unequivocal results were obtained in tests on trophoblasts cultured from chorionic villi samples obtained between 8-10 weeks of gestation. In the future, molecular genetic techniques should be able to provide early detection of the defective gene for 1,25(OH)2D3 receptor, since two-point mutations in this gene have already been described (24, 25). Amniotic fluid cells responsiveness to 1,25-(OH)2D3 reflects 1,25-(OH)2D3 action in other fetal tissues, since explants of fetal skin and kidney, obtained as early as the 17th week of gestation, and cells cultured from these tissues responded to 1,25-(OH)2D3 action with a significant increase in 25OHD3-24-hydroxylase activity. Furthermore, the inability of 1,25-(OH)2D3 to stimulate 24-hydroxylase activity in amniotic fluid cells from the affected fetus reflected the resistance to the hormone, as found after termination of pregnancy in kidney and skin tissue. In addition to presenting a practical method for the prenatal diagnosis of VDDR-II, the present study demonstrated that human fetal tissues at midgestation synthesized 24,25-(OH)2D3 and a l,25-(OH)2D3-like metabolite. Furthermore, the observation that 1,25-(OH)2D3 stimulated the in vitro synthesis of 24,25-(OH)2D3 and inhibited the synthesis of the putative 1,25-(OH)2D3 indicates that fetal tissues already have the regulatory mechanisms responsive to 1,25-(OH)2D3 that are present in differentiated tissues postnatally (26, 27). At present, however, in the absence of confirmatory structural mass spectrometric determination, the [3H]l,25-(OH)2D3-like metabolite can only be tentatively identified. The number of cells obtained from each sample of fetal tissue was too small to produce adequate amounts of the metabolite needed for such structural analysis. The accepted major role of 1,25-(OH)2D3 is to maintain calcium homeostasis (26, 27). Inasmuch as the placenta controls the influx of calcium to the fetus, it is not immediately apparent why fetal cells should require a 1,25-(OH)2D3 receptor-effector system at an early stage of gestation. Receptors for 1,25-(OH)2D3 are present, however, in various tissues that apparently are not involved in mineral metabolism (21, 26), and the hormone is a potent modulator of cell proliferation and differentiation in a variety of normal and neoplastic cells (26, 28, 29). It appears that 1,25-(OH)2D3 regulates a variety of genes involved in replication-differentiation processes, including oncogenes and genes that govern the synthesis of polyamines, cytokines, and calcium-binding proteins


WEISSMAN ET AL.

942

(26), and creatine kinase (30). Whether 1,25-(OH)2D3 is involved in the differentiation and functional development of fetal tissues by regulating the expression of these genes remains to be investigated. Interestingly, fetal skin produced the putative [3H]1,25-(OH)2D3 in vitro with a yield comparable to or even higher than that of fetal kidney tissue. The production of 1,25-(OH)2D3 by neonatal foreskin keratinocytes correlates with early events of differentiation, such as expression of transglutaminase activity and the levels of a precursor protein for the cornified envelopes, involucrine (31, 32). Production of 1,25-(OH)2D3 by keratinocytes in their early stages of growth and responsiveness to the hormone may be an autocrine mechanism by which these cells regulate epidermal and hair follicle differentiation. Whether an absence of such a regulatory mechanism in VDDR-II fetuses has a role in the pathogenesis of alopecia remains to be determined. In summary, human amniotic fluid cells and fetal tissues at midgestation, contain a responsive 1,25(OH)2D3 receptor-effector system that regulates the metabolism of 25OHD3. This receptor-mediated response to 1,25-(OH)2D3 was absent in amniotic fluid cells and tissues from a fetus with resistance to 1,25-(OH)2D3. Thus, the determination of the presence or absence of [3H]1,25-(OH)2D3 receptors and responsiveness to 1,25(OH)2D3 in amniotic fluid cells could be used for the prenatal diagnosis of VDDR type II in a high risk family. Addendum Since this manuscript was submitted for publication, we have been asked to examine amniotic fluid cells from another 23-yr-old Moslem-Arab woman, who previously gave birth to a child with VDDR-II. These cells responded to 1,25-(OH)2D3 stimulation with an 11-fold increase in 25OHD3-24-hydroxylase activity. The fetus was, therefore, judged unaffected. A normal baby girl was born. At the age of 8 months, she does not demonstrate any of the clinical or biochemical features of VDDR-II. Acknowledgment The authors thank S. Bar and Dr. Y. Nevo for expert technical assistance.

References 1. Brooks MH, Bell NH, Love L, et al. Vitamin D-dependent rickets type II: resistance of target organs to 1,25-dihydroxyvitamin D. N Engl J Med. l978;298:996-9. 2. Rosen JF, Fleishman AR, Finberg L, Hamstra A, DeLuca HF. Rickets with alopecia: an inborn error of vitamin D metabolism. J Pediatr. 1979;94:729-35. 3. Hochberg Z, Benderli A, Levi J, et al. 1,25-Dihydroxyvitamin D resistance, rickets and alopecia. Am J Med. 1984;77:805-ll. 4. Liberman UA, Eil C, Marx SJ. Resistance to 1,25-dihydroxyvitamin D association with heterogeneous defects in cultured skin

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fibroblast. J Clin Invest. 1983;73:192-200. 5. Malloy PJ, Hochberg Z, Pike WJ, Feldman D. Abnormal binding of vitamin D receptors to deoxyribonucleic acid in a kindred with vitamin D-dependent rickets, type II. J Clin Endocrinol Metab. 1989;68:263-9. 6. Castells S, Greig F, Fusi M, et al. Severely deficient binding of 1,25-dihydroxyvitamin D to its receptors in a patient responsive to high doses of this hormone. J Clin Endocrinol Metab. 1986;63:2526. 7. Marx SJ, Spiegel AM, Brown EM, et al. A familial syndrome of decrease in sensitivity to 1,25-dihydroxyvitamin D. J Clin Endocrinol Metab. 1978;47:1303-10. 8. Liberman UA, Samuel R, Halabe A, et al. End organ resistance to 1,25-dihydroxycholecalciferol. Lancet. 1980;l:504-7. 9. Takeda E, Kuroda Y, Saijo T, et al. la-Hydroxyvitamin D treatment of three patients with 1,25-dihydroxyvitamin D-receptordefect rickets and alopecia. Pediatrics. 1987;80:97-101. 10. Balsan S, Garabedian M, Larchet M, et al. Long-term nocturnal calcium infusions can cure rickets and promote normal mineralization in hereditary resistance to 1,25-dihydroxyvitamin D. J Clin Invest. 1986;77:1601-7.

11. Weisman Y, Bab I, Gazit D, Spirer Z, Jaffe M, Hochberg Z. Longterm intracaval calcium infusion therapy in end-organ resistance to 1,25-dihydroxyvitamin D. Am J Med. 1987;83:985-9. 12. Balsan S, Garabedian M, Liberman UA, et al. Rickets and alopecia with resistance to 1,25-dihydroxyvitamin D. Two different cellular defects. J Clin Endocrinol Metab. 1983;57:803-ll. 13. Feldman D, Chen T, Hirst M, Colston K, Karasek, M, Cone C. Demonstration of 1,25-dehydroxyvitamin D receptors in human skin biopsies. J Clin Endocrinol Metab. 1980;51:1463-5. 14. Eil C, Marx SJ. Nuclear uptake of 1,25-dihydroxy [3H]-cholecalciferol in dispersed fibroblasts cultured from normal human skin. Proc Natl Acad Sci USA. 1981;78:22562-6. 15. Feldman D, Chen T, Cone C, et al. Vitamin D resistant rickets with alopecia: cultured skin fibroblasts exhibit defective cytoplasmic receptors and unresponsiveness to 1,25-(OH)2D3. J Clin Endocrinol Metab. 1982;55:1020-22. 16. Griffin JE, Zerwekh JE. Impaired stimulation of 25-hydroxyvitamin D-24-hydroxylase in fibroblasts from a patient with vitamin D-dependent rickets, type II. J Clin Invest. 1983;72:1190-9. 17. Gamblin GT, Liberman UA, Eil C, Downs Jr RW, Degrange DA, Marx SJ. Vitamin D dependent rickets type II. Defective induction of 25-hydroxyvitamin D3-24-hydroxylase by 1,25-dihydroxyvitamin D3 in cultured skin fibroblasts. J Clin Invest 1985;75:954-60. 18. Virtanen I, Von Koskull H, Lehto VP, Aula P. Cultured human amniotic fluid cells characterized with antibody against intermediate filaments in indirect immunofluorescence microscopy. J Clin Invest. 1981;68:1348-55. 19. Koren R, Ravid A, Hochberg Z, Weisman Y, Novogrodsky A, Liberman UA. Defective binding and function of 1,25-dihydroxyvitamin D3 receptors in peripheral mononuclear cells of patients with end-organ resistance to 1,25-dihydroxyvitamin D3. J Clin Invest. 1985;76:2012-5. 20. Weisman Y, Harell A, Edelstein S, David M, Spirer Z, Golander A. 1,25-Dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in vitro synthesis by human decidua and placenta. Nature 1979;281:317-9. 21. Norman AW, Roth J, Orci L. The vitamin D endocrine system: steroid metabolism hormone receptors and histological response (calcium binding proteins). Endocr Rev. 1982;3:331-66. 22. Tanaka Y, DeLuca HF. Stimulation of 24,25 dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3. Science. 1975;183:1198200. 23. Delvin EE, Pilon AM, Vekemans M. Specific 1,25-hydroxy-cholecalciferol receptors and stimulation of 25-hydroxycholecalciferol24R-hydroxylase in human amniotic cells. Pediatr Res. 1987;21:423-5. 24. Hughes MR, Malloy PJ, Kieback DG, et al. Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets. Science. 1988;242:1702-5. 25. Ritchie HH, Hughes MR, Thompson ED, et al. An ochre mutation in the vitamin D receptor gene causes hereditary 1,25-dihydroxyvitamin D3-resistant rickets in three families. Proc Natl Acad Sci


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Editor-in-Chief Search Begins For Endocrine Society Journal Molecular Endocrinology The Publications Committee now solicits nominations and applications from members of the Society of candidates for the position of Editor-in-Chief of MOLECULAR ENDOCRINOLOGY. Applications will be reviewed by the Publications Committee starting October 1, 1990. All nominees will be invited to submit a curriculum vitae and a statement of interest in this position by that date. Please send nominations/applications to: Joanne S. Richards, Ph.D. Department of Cell Biology Baylor College of Medicine One Baylor Plaza Houston, TX 77030


Alopecia in patients with vitamin D-resistant rickets type-II.

Vitamin D - Dependent Rickets, Type II Case Report.

Combined use of alphafetoprotein and amniotic fluid cell morphology in early prenatal diagnosis of fetal abnormalities.

[Discolored amniotic fluid--results of prenatal diagnosis and clinical significance].

HLA typing of amniotic-fluid cells applied to prenatal diagnosis of congenital adrenal hyperplasia.

Vitamin D deficiency and rickets.

[Amniotic fluid diagnosis of fetal erythroblastosis following intrafetal transfusions (proceedings)].

Chediak-Higashi syndrome in the cat: prenatal diagnosis by evaluation of amniotic fluid cells.

25-Hydroxyvitamin D-24-hydroxylase in phytohemagglutinin-stimulated lymphocytes: intermediate bioresponse to 1,25-dihydroxyvitamin D3 of cells from parents of patients with vitamin D-dependent rickets type II.

Vitamin D-resistant rickets and 25-hydroxycholecalciferol.

Vitamin D-Deficient Rickets in Japan.

Vitamin-D deficiency rickets in Jamaican children.

Congenital rickets due to vitamin D deficiency in the mothers.

Rickets before the discovery of vitamin D.

A Practical Approach to Vitamin D Deficiency and Rickets.

Cystinotic rickets treated with vitamin D metabolites.

Fetal maturity: biochemical analyses of amniotic fluid.

Calcitriol treatment in vitamin D-dependent and vitamin D-resistant rickets.

Prenatal diagnosis of Tay-Sachs disease: studies on the reliability of hexosaminidase levels in amniotic fluid.

Rapid prenatal diagnosis of congenital Toxoplasma infection by using polymerase chain reaction and amniotic fluid.

Pterins analysis in amniotic fluid for the prenatal diagnosis of GTP cyclohydrolase deficiency.

Mutations in the vitamin D receptor and hereditary vitamin D-resistant rickets.

Novel Vitamin D Receptor Mutations in Hereditary Vitamin D Resistant Rickets in Chinese.

Normal 24-hydroxylation of vitamin D metabolites in patients with vitamin D-dependency rickets type I. Structural implications for the vitamin D hydroxylases.